To gain greater understanding of genetic control mechanisms that establish phenotypic diversity among striated myocytes, we have studied the regulation of myoglobin gene expression during embryonic development, during the transition from fetal to post-natal life, and during fast-to- slow fiber transformation induced in adult skeletal muscles by motor nerve stimulation. Work performed in the previous project period has placed us now in a position to elucidate in detail biochemical events responsible for transcriptional control of the myoglobin gene. We have defined upstream activation sequences (CCAC and A/T motifs) that function synergistically in transcriptional activation of the myoglobin gene during cardiac and skeletal muscle development. In addition, we have identified cognate proteins that bind DNA at these critical motifs. These include novel proteins (CBF40, ATF35, and MNF), as well as previously described transcription factors (MEF2, Sp1). We will address three specific aims. Specific aim (1): To identify the DNA binding proteins necessary for functional activity of the myoglobin muscle-specific enhancer. Of five protein factors present in crude myotube or cardiomyocyte extracts that bind the CCAC or A/T regions, 3 have been isolated as cDNA clones by ourselves (MNF) or others (MEF2, 5p 1), while 2 others--CBF4O and ATF35-- have not yet been characterized fully. We expect to isolate 4cDNA clones encoding these remaining proteins, and then, by a combination of gain-of- function and loss-of-function strategies, to define the relative importance of each of these 4 factors for transcriptional activity of the myoglobin gene in cardiac and skeletal myocytes. Specific aim (2): To define the molecular basis for selective expression of myoglobin in red, oxidative skeletal myofibers. Synergistic interactions between factors binding the CCAC box and A/T motifs are required for transcriptional activity of the myoglobin promoter/enhancer. We will test the hypothesis that differential expression or activity of one or more of the proteins, or of other coactivators that form protein-protein contacts with these DNA-binding proteins, provides the basis for the selective expression of myoglobin in Type I and Type IIa myofibers. Specific aim (3): To define the functional roles of MNFs in muscle development. Our working hypothesis is that MNFs are physiological regulators of myoglobin gene expression. We are eager, however, to define other functions of these novel proteins. We propose to define spatial and temporal patterns of expression of MNFs during fetal and post-natal development, to disrupt the MNF gene and to define the resulting phenotype, to define consensus binding sites for MNFs, which should lead to the identification of other genes activated by MNF. These studies, in aggregate, have excellent potential for defining molecular details of myoglobin enhancer function at high resolution, and provide opportunities to define the functions of novel and interesting transcription factors. We expect these experiments to elucidate general mechanisms by which the distinctive phenotypes of red and white skeletal myofibers are established and maintained.